Abstract:

The present invention is to provide a process for efficiently producing an
optically active alcohol including (R)-3-hydroxy-3-phenylpropanenitrile.
One of the features of the present invention is a polypeptide having an
activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile isolated
from a microorganism belonging to the genus Candida to produce
(R)-3-hydroxy-3-phenylpropanenitrile, DNA encoding the polypeptide and a
transformant of producing the polypeptide. Another feature of the present
invention is a process for producing an optically active alcohol such as
(R)-3-hydroxy-3-phenylpropanenitrile by reducing a carbonyl compound such
as 3-oxo-3-phenylpropanenitrile by use of the polypeptide or the
transformant.

Claims:

1. DNA represented by the following (a) or (b)(a) DNA containing a base
sequence represented by SEQ ID NO: 1 of a sequence listing(b) DNA capable
of hybridizing with DNA consisting of a complementary base sequence to
the base sequence represented by SEQ ID NO: 1 of the sequence listing
under stringent conditions, and encoding a polypeptide having an activity
of asymmetrically reducing 3-oxo-3-phenylpropanenitrile represented by
the formula (1) below: ##STR00011## to produce
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
below: ##STR00012##

3. A polypeptide encoded by DNA according to claim 1 and having an
activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile
represented by the formula (1) above to produce
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
above.

4. A polypeptide consisting of an amino acid sequence represented by SEQ
ID NO: 2 of the sequence listing.

5. A vector comprising DNA according to claim 1.

6. A vector according to claim 5 being plasmid pNTCM.

7. The vector according to claim 5, further comprising DNA encoding a
polypeptide having an activity of glucose dehydrogenase.

8. The vector according to claim 7, wherein the polypeptide having an
activity of glucose dehydrogenase is glucose dehydrogenase derived from
Bacillus megaterium.

9. A transformant obtained by transforming a host cell with the vector
according to claim 5.

10. The transformant according to claim 9, wherein the host cell is
Escherichia coli.

12. A transformant to which both DNA according to claim 1 and DNA encoding
a polypeptide having an activity of glucose dehydrogenase are introduced.

13. A process for producing an optically active alcohol characterized by
reacting the polypeptide according to claim 3, a transformant obtained by
transforming a host cell with a vector comprising DNA represented by the
following (a) or (b)(a) DNA containing a base sequence represented by SEQ
ID NO: 1 of a sequence listing(b) DNA capable of hybridizing with DNA
consisting of a complementary base sequence to the base sequence
represented by SEQ ID NO: 1 of the sequence listing under stringent
conditions, and encoding a polypeptide having an activity of
asymmetrically reducing 3-oxo-3-phenylpropanenitrile represented by the
formula (1) below: ##STR00013## to produce
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
below: ##STR00014## or a cultured product of the transformant with a
compound having a carbonyl group.

14. The process according to claim 13, wherein the compound having a
carbonyl group is 3-oxo-3-phenylpropanenitrile represented by the formula
(1) above and the optically active alcohol is
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
above.

15. The process according to claim 13, wherein the compound having a
carbonyl group is ethyl 4-chloro-3-oxobutyrate represented by the formula
(3) below: ##STR00015## and the optically active alcohol is ethyl
(S)-4-chloro-3-hydroxybutyrate represented by the formula (4) below:
##STR00016##

16. The process according to claim 13, wherein the compound having a
carbonyl group is 2-chloro-1-(3'-chlorophenyl)ethanone represented by the
formula (5) below: ##STR00017## and the optically active alcohol is
(R)-2-chloro-1-(3'-chlorophenyl)ethanol represented by the formula (6)
below: ##STR00018##

17. The process according to claim 13, wherein the compound having a
carbonyl group is tert-butyl (S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate
represented by the formula (7) below: ##STR00019## and the optically
active alcohol is tert-butyl (3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoate
represented by the formula (8) below: ##STR00020##

Description:

TECHNICAL FIELD

[0001]The present invention relates to a polypeptide (carbonyl reductase)
having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile represented by the formula (1) below:

##STR00001##

to produce (R)-3-hydroxy-3-phenylpropanenitrile represented by the formula
(2) below:

##STR00002##

and isolated from a microorganism having said activity; DNA encoding the
polypeptide; a vector containing the DNA; and a transformant transformed
with the vector.

[0002]The present invention also relates to a process for producing an
optically active alcohol, in particular,
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2) above
using the polypeptide or the transformant.

[0003](R)-3-hydroxy-3-phenylpropanenitrile is a useful compound as an
intermediate of a pharmaceutical product such as a β-adrenaline
receptor blocker.

[0004]The entire disclosure including the specification, claims, drawings
and abstract of Japanese Patent No. 2004-312365 (filed Oct. 27, 2004) is
incorporated herein by references in its entirety.

BACKGROUND ART

[0005]As a process for producing optically active
3-hydroxy-3-phenylpropanenitrile, there are known 1) a process for
deriving it from an optically active precursor substance (Patent
Documents 1 and 2); 2) a process for optically resolving racemic
3-hydroxy-3-phenylpropanenitrile with a hydrolyzing enzyme such as lipase
(Patent Document 3, Non-Patent Document 2); 3) a process for condensing
benzaldehyde and acetonitrile in the presence of an asymmetric ligand and
a metallic catalyst (Non-patent document 3); 4) a process for reducing an
carbonyl group of 3-oxo-3-phenylpropanenitrile in the presence of an
asymmetric ligand or an asymmetric catalyst (Patent Document 4 and
Non-Patent Document 4); and 5) a process for reducing an carbonyl group
of 3-oxo-3-phenylpropanenitrile by use of a microorganism (Non-Patent
Documents 5 and 6).

[0016]The processes disclosed in the aforementioned documents individually
have drawbacks. In the process 1), an expensive precursor substance is
required. In the process 2), since a theoretical yield is 50%, a half of
a raw material is wasted. In the processes 3) and 4), an expensive
asymmetric ligand or asymmetric catalyst is required. In the process 5),
a productivity per reaction volume is low. Any one of these processes is
far from an economic process.

[0017]In view of the aforementioned circumstances, an object of the
present invention is to provide a useful polypeptide in producing
(R)-3-hydroxy-3-phenylpropanenitrile, DNA encoding the polypeptide, a
vector containing the DNA, and a transformant transformed with the
vector.

[0018]Another object of the present invention is to provide processes for
effectively producing various types of optically active alcohols
including (R)-3-hydroxy-3-phenylpropanenitrile, by use of the polypeptide
and the transformant.

[0019]The present inventors isolated a polypeptide having an activity of
asymmetrically reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile from a microorganism having the
activity. Furthermore, they isolated DNA encoding the polypeptide and
succeeded in creating a transformant capable of producing the polypeptide
by using the DNA in high yield. Moreover, they found that various types
of useful optically active alcohols including
(R)-3-hydroxy-3-phenylpropanenitrile can be efficiently produced by use
of the polypeptide or the transformant. Based on these, the present
invention was accomplished.

[0020]More specifically, one of the features of the present invention is a
polypeptide having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile. Another feature of the present
invention is DNA encoding the polypeptide. Still another feature of the
present invention is a vector containing the DNA. A further feature of
the present invention is a transformant containing the vector. Still a
further feature of the present invention is a process for producing an
optically active alcohol including (R)-3-hydroxy-3-phenylpropanenitrile,
by use of the polypeptide or the transformant.

[0021]The present invention can provide a practical process for producing
a useful and optically active alcohol including
(R)-3-hydroxy-3-phenylpropanenitrile.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 shows the method and structure of a recombinant vector
pNTCMG1.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023]The present invention will be more specifically explained below.

[0024]Gene manipulation such as isolation of DNA, preparation of a vector,
and formation of a transformant can be performed in accordance with the
method described in publications such as Molecular Cloning 2nd Edition
(Cold Spring Harbor Laboratory Press, 1989), unless otherwise specified.
Furthermore, the reference symbol "%" used in the description of the
specification refers to % (w/v), unless otherwise specified.

[0025]1. Polypeptide and its Source

[0026]The polypeptide of the present invention is one having an activity
of asymmetrically reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile. Such a polypeptide can be isolated
from a microorganism having the activity. The microorganism from which
the polypeptide of the present invention is derived is not particularly
limited. For example, yeast belonging to the genus Candida may be
mentioned. Particularly preferably, Candida magnoliae strain NBRC0661 may
be mentioned. The microorganism is available from an independent
organization, the National Institute of Technology and Evaluation,
Biological Resource Center (NBRC, 2-5-8 Kazusakamatari, Kisarazu-shi,
Chiba, 292-0818, JAPAN).

[0027]As a medium for culturing a microorganism from which the polypeptide
of the present invention is derived, any liquid nutrition medium
generally used and containing a carbon source, nitrogen source, inorganic
salts, and organic nutrients, etc., can be used as long as the
microorganism can be proliferated in the medium.

[0028]2. Isolation of Polypeptide

[0029]Isolation of the polypeptide of the present invention from a
microorganism from which the polypeptide is derived can be generally
performed by using known protein purification methods in appropriate
combination, for example, as described below. First, the microorganism is
cultured in an appropriate medium and microorganism's cells are collected
from the culture solution by centrifugation or filtration. The obtained
cells are disrupted by an ultrasonic homogenizer or a physical means
using glass beads, and thereafter, cell residues are centrifugally
removed to obtain a cell-free extract. Subsequently, the cell-free
extract is subjected to a process such as salting-out (e.g.,
precipitation with ammonium sulfate and precipitation with sodium
phosphate, etc.), precipitation with a solvent (precipitation of a
protein fraction by acetone, ethanol or the like), dialysis, gel
filtration chromatography, ion exchange chromatography, reversed-phase
chromatography, and ultrafiltration, singly or in combination. In this
manner, the polypeptide of the present invention is isolated from the
cell-free extract.

[0030]3. Reduction Reaction

[0031]The activity of reducing 3-oxo-3-phenylpropanenitrile can be
determined, for example, by adding a 0.5 mM 3-oxo-3-phenylpropanenitrile
as a substrate, 0.25 mM coenzyme NADPH and a crude enzyme to a 100 mM
phosphate buffer (pH 6.5) containing 0.33% (v/v) dimethylsulfoxide,
allowing the mixture to react at 30° C. for one minute, measuring
absorbance of the resultant reaction solution at a wavelength of 340 nm,
and calculating the activity from the reduction rate of the absorbance.

[0034]The DNA of the present invention is one encoding the polypeptide of
the present invention mentioned above. Any DNA may be used as long as it
can express the polypeptide in a host cell to which the DNA is introduced
in accordance with a method as described later. The DNA may optionally
contain a non-translation region. As long as the polypeptide can be
obtained, one skilled in the art can obtain such DNA from a microorganism
from which the polypeptide is derived, in accordance with a known method,
for example, the method set forth below.

[0035]First, the isolated polypeptide of the present invention is digested
with an appropriate endopeptidase. The resultant peptide fragments are
separated by reverse-phase HPLC. Subsequently, the whole or part of amino
acid sequences of these peptide fragments are determined, for example, by
an ABI492-type protein sequencer (manufactured by Applied Biosystems).

[0036]Based on the amino acid sequence data thus obtained, a PCR
(Polymerase Chain Reaction) primer is synthesized for amplifying a part
of DNA encoding the polypeptide. Subsequently, the chromosomal DNA of a
microorganism from which the polypeptide is derived is prepared by a
general DNA isolation method, for example, a method of Vissers et al.
(Appl. Microbiol. Biotechnol., 53, 415 (2000)). PCR is performed using
the chromosomal DNA as a template and the PCR primer described above to
amplify a part of the DNA encoding the polypeptide and the base sequence
thereof is determined. The determination of the base sequence can be
performed for example, by an ABI373-type DNA Sequencer (manufactured by
Applied Biosystems).

[0037]When a part of DNA encoding the polypeptide is clarified, the whole
sequence of the DNA can be determined by use of, for example, i-PCR
method (Nucl. Acids Res., 16, 8186 (1988)).

[0038]As an example of DNA of the present invention thus obtained, mention
may be made of DNA containing a base sequence represented by Sequence No.
1 of the sequence listing. Furthermore, DNA that is hybridized, under
stringent conditions, with the DNA having a base sequence represented by
Sequence No. 1 of the sequence listing and a complementary base sequence
thereof and that is encoding a polypeptide having an activity of
asymmetrically reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile may be also included in the DNA of
the present invention.

[0039]DNA that is hybridized, under stringent conditions, with the DNA
having a base sequence represented by Sequence No. 1 of the sequence
listing and a complementary base sequence thereof refers to one that
specifically forms a hybrid with the DNA having a base sequence
represented by Sequence No. 1 of the sequence listing and a complementary
base sequence thereof when a colony hybridization method, plaque
hybridization method, Southern hybridization method or the like is
performed.

[0040]The stringent conditions herein refer to those in which
hybridization is performed at 65° C. in an aqueous solution having
a composition of 75 mM trisodium citrate, 750 mM sodium chloride, 0.5%
sodium dodecyl sulfate, 0.1% bovine serum albumin, 0.1%
polyvinylpyrrolidone and 0.1% Ficoll 400 (manufactured by Amersham
Bioscience), followed by washing with an aqueous solution having a
composition of 15 mM trisodium citrate, 150 mM sodium chloride, and 0.1%
sodium dodecyl sulfate at 60° C. Preferably, after hybridization
is performed in the aforementioned conditions, washing is performed by
use of an aqueous solution having a composition of 15 mM trisodium
citrate, 150 mM sodium chloride, and 0.1% sodium dodecyl sulfate, at
65° C., and more preferably, washing is performed with an aqueous
solution containing 1.5 mM trisodium citrate, 15 mM sodium chloride, and
0.1% sodium dodecyl sulfate, at 65° C.

[0041]5. Polypeptide

[0042]As an example of the polypeptide of the present invention, mention
can be made of a polypeptide consisting of the amino acid sequence
represented by Sequence No. 2 of the sequence listing and encoded by the
base sequence represented by Sequence No. 1 of the sequence listing.

[0043]Furthermore, the polypeptide having a homology of not less than a
certain value with the polypeptide consisting of the amino acid sequence
represented by Sequence No. 2 of the sequence listing, and having an
activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile to
produce (R)-3-hydroxy-3-phenylpropanenitrile is functionally equivalent
to the polypeptide and included in the present invention.

[0044]The homology of the sequence herein is expressed, for example, by an
identity value relative to the entire sequence when two amino acid
sequences are analyzed and compared by a homology search program FASTA
(W. R. Pearson & D. J. Lipman P.N.A.S. (1988) 85:2444-2448). As a
polypeptide equivalent to that consisting of the amino acid sequence
represented by Sequence No. 2 of the sequence listing, mention may be
made of a polypeptide having a homology of 70% or more to the
polypeptide, preferably 80% or more, and more preferably 90% or more.

[0045]Such a polypeptide can be obtained by, for example, ligating DNA,
which hybridizes, under stringent conditions, with the DNA having a base
sequence represented by Sequence No. 1 of the sequence listing and a
complementary base sequence thereof as previously described, to an
appropriate vector, introducing into an appropriate host cell, and
expressing the vector. Alternatively, such a polypeptide can be also
obtained by processing the polypeptide consisting of amino acid sequence
represented by Sequence No. 2 of the sequence listing to substitute,
insert, delete or add an amino acid(s) in accordance with known methods
described, for example, in Current Protocols in Molecular Biology (John
Wiley and Sons, Inc., 1989). The number of amino acids to be replaced,
inserted, deleted or added in the aforementioned polypeptide is not
particularly limited as long as the activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile is not significantly decreased;
however, the number of amino acids is 20 amino acids or less, preferably
5 or less, and further preferably 2 or 1.

[0046]6. Vector and Transformant

[0047]The vector for use in introducing the DNA of the present invention
into a microbial host and expressing the DNA introduced in the microbial
host is not particularly limited as long as the gene encoded by the DNA
can be expressed in an appropriate microbial host. Examples of such a
vector include a plasmid vector, phage vector and cosmid vector. Besides
these, a shuttle vector capable of exchanging a gene with another host
strain may be used.

[0048]Such a vector generally contains regulators such as lac UV5
promoter, trp promoter, trc promoter, tac promoter, lpp promoter, tufB
promoter, recA promoter, and pL promoter and can be suitably used as an
expression vector containing an expression unit operably linked with the
DNA of the present invention. For example, pUCNT (WO 94/03613) can be
suitably used.

[0049]The term "regulatory element" used in the specification refers to a
base sequence having a functional promoter and any of transcription
elements (such as an enhancer, CCAAT box, TATA box, and SPI site).

[0050]The term "operably linked" used in the specification means that
various regulatory elements such as a promoter and an enhancer for
regulating the expression of a gene are ligated to the gene in a
functional state in a host cell. It is known to one skilled in the art
that the type and kind of regulatory element may vary depending upon the
host.

[0051]Examples of the expression vector of the present invention include
pNTCM (described later) having the DNA represented by Sequence No. 1
introduced in pUCNT.

[0052]As a host cell to which a vector containing the DNA of the present
invention is introduced, mention may be made of bacterial cells, yeasts,
fungi, vegetable cells and animal cells. In view of introduction and
expression efficiencies, bacterial cells, in particular, Escherichia
coli, is preferable. The vector containing the DNA of the present
invention can be introduced into a host cell by a known method. When E.
coli is used as a host cell, the vector can be introduced into the host
cell by using, for example, a commercially available E. coli HB101
competent cell (manufactured by Takara Bid Inc.).

[0053]As an example of a transformant according to the present invention,
mention may be made of E. coli HB101 (pNTCM) FERM BP-10418 (described
later). The transformant has been deposited as of Sep. 26, 2005 under the
aforementioned accession number with an independent organization, the
International Patent Organism Depositary of the National Institute of
Advanced Industrial Science and Technology (located at Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan). Note that the original
national deposition date of Oct. 23, 2003 for the strain has been
transferred to international deposition based on the Budapest Treaty.

[0054]7. Polypeptide Having a Coenzyme Regeneration Activity

[0055]When the polypeptide of the present invention is reacted in contact
with a compound having a carbonyl group and a coenzyme such as NADPH, it
asymmetrically reduces the compound having a carbonyl group to produce an
optically active alcohol. At this time, as the reaction proceeds, a
coenzyme such as NADPH is converted into an oxidation type. However, when
the reaction of the polypeptide of the present invention is performed in
the presence of a polypeptide having an ability (hereinafter referred to
as "coenzyme regeneration ability") to convert the oxidation-type
coenzyme into a reduction type and the compound serves as a substrate for
the polypeptide, the amount of an expensive coenzyme can be greatly
reduced. Examples of the polypeptide having a coenzyme regeneration
ability include hydrogenase, formic acid dehydrogenase, alcohol
dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase
and glucose dehydrogenase. Preferably, glucose dehydrogenase may be used.

[0056]8. Use of Transformant

[0057]When a transformant containing DNA encoding the polypeptide is used
in place of the polypeptide of the present invention, an optically active
alcohol can be also produced. Furthermore, even if a transformant
containing both DNA encoding the polypeptide of the present invention and
DNA encoding the polypeptide having a coenzyme regeneration ability is
used, an optically active alcohol can be also produced. In particular,
when a transformant containing both DNA encoding the polypeptide of the
present invention and DNA encoding the polypeptide having a coenzyme
regeneration ability is used, an optically active alcohol can be produced
more effectively because it is not necessary to separately prepare/add an
enzyme for regenerating the coenzyme.

[0058]Note that, with respect to a transformant containing DNA encoding
the polypeptide of the present invention or a transformant containing
both DNA encoding the polypeptide of the present invention and DNA
encoding the polypeptide having a coenzyme regeneration ability, not only
cultured bacterial cells of the transformant but also a processed product
of the transformant can be used to produce an optically active alcohol.
The "processed product of the transformant" refers to cells treated with
a surfactant and an organic solvent, dried cells, disrupted cells, crude
cell-extract or a mixture thereof. Besides these, it refers to
immobilized cells fixed by means of a known means such as a crosslinking
method, physical adsorption method and entrapment method.

[0059]The transformant containing both DNA encoding the polypeptide of the
present invention and DNA encoding the polypeptide having a coenzyme
regeneration ability can be obtained by introducing both DNA encoding the
polypeptide of the present invention and DNA encoding the polypeptide
having a coenzyme regeneration ability into a single vector and inserting
the vector to a host cell. Or it may be also obtained by introducing
these two types of DNA fragments into two different types of vectors of a
heterothallism group, respectively, and inserting these two types of
vectors to a single host cell.

[0060]As an example of a vector into which both DNA encoding the
polypeptide of the present invention and DNA encoding a polypeptide
having a coenzyme regeneration ability are introduced, mention may be
made of pNTCMG1 (described later), which is the aforementioned expression
vector pNTCM having a glucose dehydrogenase gene derived from Bacillus
megaterium introduced therein. Furthermore, as an example of a
transformant containing both DNA encoding the polypeptide of the present
invention and DNA encoding a polypeptide having a coenzyme regeneration
ability, mention may be made of E. coli HB101 (pNTCMG1) (described later)
obtained by transforming E. coli HB101 with the vector.

[0061]Culturing a transformant containing DNA encoding the polypeptide of
the present invention and a transformant containing both DNA encoding the
polypeptide of the present invention and DNA encoding the polypeptide
having a coenzyme regeneration ability can be performed in a liquid
nutrition medium generally used and containing a carbon source, nitrogen
source, inorganic salts and organic nutrients, etc., as long as they are
proliferated.

[0062]The activity of the polypeptide having a coenzyme regeneration
activity in a transformant can be measured by a conventional method. For
example, the activity of glucose dehydrogenase can be obtained by adding
100 mM glucose, 2 mM coenzyme NADP or NAD and an enzyme to a 1M
tris-hydrochloric acid buffer (pH 8.0) and allowing the reaction mixture
to react at 25° C. for one minute, measuring absorbance at a
wavelength of 340 nm, and calculating an increasing rate of the
absorbance.

[0063]9. Production of Optically Active Alcohol

[0064]Production of an optically active alcohol using either the
polypeptide of the present invention or a transformant containing DNA
encoding the polypeptide can be performed by adding a compound containing
a carbonyl group serving as a substrate, a coenzyme such as NADPH and the
polypeptide of the present invention or the transformant containing DNA
encoding the polypeptide to an appropriate solvent, and stirring the
mixture while adjusting the pH.

[0065]On the other hand, when a reaction is performed using the
polypeptide of the present invention in combination with a polypeptide
having a coenzyme regeneration ability, the polypeptide having a coenzyme
regeneration ability (e.g., glucose dehydrogenase) and a compound serving
as a substrate thereof (e.g., glucose) are further added to the reaction
composition mentioned above. Note that, also in the latter case, when the
transformant containing both DNA encoding the polypeptide of the present
invention and DNA encoding the polypeptide (e.g., glucose dehydrogenase)
having a coenzyme regeneration ability or a processed product thereof is
used, it is not necessary to separately add the polypeptide (e.g.,
glucose dehydrogenase) having a coenzyme regeneration ability.

[0066]The reaction may be performed in an aqueous solvent or in a mixture
of an aqueous solvent and an organic solvent. Examples of the organic
solvent include toluene, ethyl acetate, n-butyl acetate, hexane,
isopropanol, diisopropyl ether, methanol, acetone, and dimethylsulfoxide.
The reaction may be performed at a temperature of 10° C. to
70° C. and the pH of the reaction solution is maintained at 4 to
10. The reaction is performed in a batch system or a continuous system.
In the case of the batch system, a reaction substrate is added in an
initial concentration of 0.1% to 70% a (w/v).

[0067]10. Substrate and Product

[0068]Examples of the compound having a carbonyl group serving as a
substrate, include 3-oxo-3-phenylpropanenitrile represented by, for
example, the formula (1) below:

[0072]However, the compound containing a carbonyl group is not
particularly limited as long as it can be reduced in the aforementioned
reaction conditions and converted into an optically active alcohol.

[0073]In the aforementioned reaction conditions, when
3-oxo-3-phenylpropanenitrile represented by the formula (1) above is used
as a substrate, (R)-3-hydroxy-3-phenylpropenenitrile represented by the
formula (2) below:

##STR00007##

can be obtained.

[0074]When ethyl 4-chloro-3-oxobutyrate represented by the formula (3)
above is used as a substrate, ethyl (S)-4-chloro-3-hydroxybutyrate
represented by the formula (4) below:

##STR00008##

can be obtained.

[0075]When 2-chloro-1-(3'-chlorophenyl)ethanone represented by the formula
(5) above is used as a substrate, (R)-2-chloro-1-(3'-chlorophenyl)ethanol
represented by the formula (6) below:

##STR00009##

can be obtained.

[0076]Furthermore, when tert-butyl
(S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate represented by the formula (7)
above is used as a substrate, tert-butyl
(3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoate represented by the formula
(8) below:

##STR00010##

can be obtained.

[0077]11. Purification and Analysis

[0078]An optically active alcohol produced by the reaction can be purified
by a conventional method, for example, by extracting a reaction solution
containing an optically active alcohol produced by the reaction with an
organic solvent such as ethyl acetate and toluene, removing the organic
solvent by distillation under reduced pressure, and subjecting the
resultant mixture to distillation, recrystallization or chromatographic
process.

[0083]As described in the foregoing, according to the present invention,
the polypeptide of the present invention can be efficiently produced and
a process for producing a useful and optically active alcohol represented
by (R)-3-hydroxy-3-phenylpropanenitrile can be provided by use of the
polypeptide.

EXAMPLES

[0084]The present invention will be more specifically described by way of
Examples below, which will not be construed as limiting the invention.
Note that specific manipulation methods regarding recombinant DNA
techniques used in the following Examples are described in the following
publications:

[0087]In accordance with the following method, a polypeptide from Candida
magnoliae strain NBRC0661, having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile was separated and purified as a
single product. Purification operation was performed at 4° C.
unless otherwise specified.

[0088]The reduction activity to 3-oxo-3-phenylpropanenitrile was obtained
by dissolving a substrate, 3-oxo-3-phenylpropanenitrile so as to have a
final concentration of 0.5 mM and a coenzyme NADPH in a final
concentration of 0.25 mM in a 100 mM phosphate buffer (pH 6.5) containing
0.33% (v/v) of dimethylsulfoxide, further adding a crude enzyme solution,
reacting the mixture at 30° C. for one minute, measuring
absorbance of the reaction solution at a wavelength of 340 nm, and
calculating a reduction rate of the absorbance. In the reaction
conditions, the activity of oxidizing 1 μmol of NADPH to NADP per
minute was defined as 1 unit.

[0091]To this medium, 180 ml of a culture solution of Candida magnoliae
strain NBRC0661 previously cultured in the same type of medium was
inoculated. The culture medium was cultured with stirring at a rotation
rate of 250 rpm and supplying air at a rate of 5.0 NL/min at 30°
C. and while adjusting the lower pH value to 5.5 by adding a 30% (w/w)
aqueous sodium hydroxide solution dropwise. Culturing was performed for
30 hours, while adding 655 g of a 55% (w/w) aqueous glucose solution each
of 18 hours, 22 hours and 26 hours after initiation of culturing.

[0092](Preparation of Cell-Free Extraction)

[0093]Bacterial cells were centrifugally collected from the culture
solution above and washed with a 0.8% aqueous sodium chloride solution.
The bacterial cells were suspended in a 100 mM phosphate buffer (pH 7.0)
containing 5 mM of β-mercaptoethanol and 0.1 mM phenyl methane
sulfonyl fluoride and disrupted by a Dyno-mill (manufactured by Willy A.
Bachofen AG.). Thereafter, cell residues were centrifugally removed from
the disrupted material to obtain 1900 ml of a cell-free extract.

[0094](Removal of Nucleic Acid)

[0095]To the cell-free extract obtained above, 100 ml of a 5% aqueous
protamine sulfate solution was added. The mixture was stirred overnight
and centrifuged to remove a precipitate.

[0096](Fractionation with Ammonium Sulfate)

[0097]After nucleic acids were removed in the above, the pH of the crude
enzyme solution was adjusted to 7.0 by ammonia water. While maintaining
the pH, ammonium sulfate was added and dissolved so as to obtain 50%
saturation. The precipitate generated was centrifugally removed. To the
supernatant, ammonium sulfate was further added and dissolved so as to
obtain 60% saturation while maintaining the pH to 7.0 in the same manner
as above. The generated precipitate was centrifugally collected. The
precipitate was dissolved in a 10 mM phosphate buffer (pH 7.0) and
dialyzed against the same buffer overnight to obtain active fractions.

[0098](DEAE TOYOPEARL Column Chromatography)

[0099]The active fractions obtained by fractionation with ammonium sulfate
were loaded to a DEAE-TOYOPEARL 650M column (400 ml, manufactured by
TOSOH Corporation), which was previously equilibrated with a 10 mM
phosphate buffer (pH 7.0) to adsorb the active fractions. After the
column was washed with the same buffer, the active fractions were eluted
by linear gradient with NaCl (0 M to 0.5 M). The active fractions were
collected and dialyzed against a 10 mM phosphate buffer (pH 7.0)
overnight.

[0100](Blue Sepharose Column Chromatography)

[0101]The active fractions obtained by DEAE-TOYOPEARL column
chromatography were loaded to Blue Sepharose CL-6B column (50 ml,
manufactured by Amersham Biosciences), which was previously equilibrated
with a 10 mM phosphate buffer (pH 7.0) to adsorb the active fractions.
After the column was washed with the same buffer, the active fractions
were eluted by linear gradient with NaCl (0 M to 1 M). The active
fractions were collected and dialyzed against a 10 mM phosphate buffer
(pH 7.0) overnight.

[0102](2',5'-ADP Sepharose Column Chromatography)

[0103]The active fractions obtained by Blue sepharose column
chromatography were loaded to 2',5'-ADP sepharose column (20 ml,
manufactured by Amersham Biosciences), which was previously equilibrated
with a 10 mM phosphate buffer (pH 7.0) to adsorb the active fractions.
After the column was washed with the same buffer, the active fractions
were eluted by linear gradient with NaCl (0 M to 1 M). The active
fractions were collected and dialyzed against a 10 mM phosphate buffer
(pH 7.0) overnight.

[0104](HiTrap Phenyl HP Column Chromatography)

[0105]To the active fractions obtained by 2',5'-ADP sepharose column
chromatography, ammonium sulfate was added so as to obtain a final
concentration of 35%. The mixture was loaded to a HiTrap Phenyl HP column
(manufactured by Amersham Biosciences), which was previously equilibrated
with a 10 mM phosphate buffer (pH 7.0) containing 35% ammonium sulfate,
to adsorb the active fractions. After the column was washed with the same
buffer, the active fractions were eluted by linear gradient with NaCl
(35% to 0%). The active fractions were collected and dialyzed against a
10 mM phosphate buffer (pH 7.0) overnight to obtain a purified
polypeptide sample.

Example 2

Gene Cloning

[0106](Preparation of PCR Primer)

[0107]The purified polypeptide obtained in Example 1 was denatured in the
presence of 8M urea, and thereafter digested with lysyl end-peptidase
(manufactured by Wako Pure Chemical Industries Ltd.) derived from
Achromobacter. The amino acid sequences of the obtained peptide fragments
were determined by an ABI492-type protein sequencer (manufactured by
PerkinElmer). Based on the putative DNA sequence from the amino acid
sequence, a primer 1: 5'-cargarcaytaygtntggcc-3'(Sequence No. 3 of the
sequence listing) and primer 2: 5'-atygcrtcnggrtadatcca-3' (Sequence No.
4 of the sequence listing) were synthesized for amplifying a part of the
gene encoding the polypeptide by PCR.

[0108](PCR Amplification of Gene)

[0109]Chromosomal DNA was extracted from bacterial cells of Candida
magnoliae strain NBRC0661 cultured in the same manner as in Example 1 in
accordance with the method of Visser et al. (Appl. Microbiol.
Biotechnol., 53, 415 (2000)). Subsequently, PCR was performed using the
DNA primers 1 and 2 prepared above, and the chromosomal DNA thus obtained
as a template. As a result, a DNA fragment of about 0.5 kbp, which was
conceivably a part of the desired gene, was amplified. PCR was performed
by using TaKaRa Ex Taq (manufactured by Takara Bio Inc.) as DNA
polymerase under reaction conditions according to the instructions. The
DNA fragment was cloned to plasmid pT7Blue T-Vector (manufactured by
Novagen) and the base sequence thereof was analyzed by use of ABI PRISM
Dye Terminator Cycle Sequencing Ready Reaction Kit (manufactured by
PerkinElmer) and an ABI 373A DNA Sequencer (manufactured by PerkinElmer).
The resultant base sequence is shown in Sequence No. 5 of the sequence
listing.

[0110](Determination of the Entire Sequence of Desired Gene by i-PCR
Method)

[0111]The chromosomal DNA of Candida magnoliae strain NBRC0661 prepared
above was completely digested with restriction enzyme PstI. A mixture of
the obtained DNA fragments was treated with T4 ligase to form
intramolecular cyclization. Using this as a template, i-PCR method (Nucl.
Acids Res., 16, 8186 (1988)) was performed. In this manner, the entire
base sequence of a gene containing the base sequence represented by
Sequence No. 5 above. The results are shown in Sequence No. 1 of the
sequence listing. The aforementioned i-PCR was performed using TaKaRa LA
Taq (Takara Bio. Inc.) as DNA polymerase under the reaction conditions
according to the instructions. The amino acid sequence encoded by the
base sequence represented by Sequence No. 1 is shown in Sequence No. 2.

Example 3

Construction of Expression Vector

[0112]PCR was performed using primer 3:
5'-gtgcatatgtcttctcttcacgctcttg-3'(Sequence No. 6 of the sequence
listing) and primer 4: 5'-ggcgaattcttattaaacggtagagccattgtcg-3'(Sequence
No. 7 of the sequence listing), and chromosomal DNA of Candida magnoliae
strain NBRC0661 obtained in Example 2 as a template. As a result, a
double stranded DNA was obtained having an NdeI recognition site added to
the portion of the initiation codon of a gene consisting of the base
sequence represented by Sequence No. 1 of the sequence listing and an
EcoRI recognition site added immediately after the termination codon. PCR
was performed using TaKaRa LA Taq (manufactured by Takara Bio Inc.) as
DNA polymerase under the reaction conditions according to the
instructions. The DNA was digested with NdeI and EcoRI and inserted
between the NdeI recognition site and the EcoRI recognition site
downstream of the lac promoter of plasmid pUCNT (WO 94/03613). In this
manner, a recombination vector pNTCM was constructed.

[0113]PCR was performed using primer 5:
5'-gccgaattctaaggaggttaacaatgtataaa-3'(Sequence No. 8 of the sequence
listing) and primer 6: 3'-gcggtcgacttatccgcgtcctgcttgg-5'(Sequence No. 9
of the sequence listing), and plasmid pGDK1 (Eur. J. Biochem., 186, 389
(1989)) as a template. As a result, a double stranded DNA was obtained in
which a ribosome connecting sequence of E. coli was added to a position
by 5 bases upstream of the initiation codon of a glucose dehydrogenase
gene (hereinafter referred to as "GDH") derived from Bacillus megaterium
strain LAM1030, further an EcoRI cleaving site was added to the site
immediately therebefore, and an SalI cleaving site was added to
immediately after the termination codon. The DNA fragment thus obtained
was digested with EcoRI and SalI and inserted between the EcoRI
recognition site and the SalI recognition site downstream of the lac
promoter of plasmid pUCNT (WO 94/03613). In this manner, a recombination
vector pNTG1 was constructed.

[0114]Subsequently, a double stranded DNA was prepared having an NdeI
recognition site added to the portion of the initiation codon of a gene
consisting of the base sequence represented by Sequence No. 1 of the
sequence listing and an EcoRI recognition site added to the portion
immediately after the termination codon, in the same manner as in Example
3. The resultant DNA was inserted between the NdeI recognition site and
the EcoRI recognition site of the recombinant vector pNTG1 mentioned
above. In this manner, a recombinant plasmid pNTCMG1 was constructed. The
preparation method and structure of pNTCMG1 are shown in FIG. 1.

Example 5

Preparation of Transformant

[0115]Using the recombinant vector pNTCM constructed in Example 3, the E.
coli HB101 competent cell (manufactured by Takara Bio Inc.) was
transformed to obtain E. coli HB101 (pNTCM). This transformant has been
deposited as of Sep. 26, 2005 under the accession number of FERM BP-10418
at an independent organization, the International Patent Organism
Depositary of the National Institute of Advanced Industrial Science and
Technology (located at Tsukuba Central 6, 1-1-1 Higashi, Tsukuba,
Ibaraki, 305-8566, Japan). Note that the original national deposition
date of Oct. 23, 2003 for the strain has been transferred to
international deposition based on the Budapest Treaty.

[0117]Two types of transformants obtained in Example 5, and E. coli HB101
(pUCNT), which is a transformant containing a vector plasmid pUCNT, were
separately inoculated in 50 ml of 2×YT medium (1.6% of tryptone,
1.0% of yeast extract, 0.5% of NaCl, pH 7.0) containing 200 μg/ml
ampicillin and cultured while shaking at 37° C. for 24 hours.
Bacterial cells were centrifugally collected and suspended in 50 ml of
100 mM phosphate buffer (pH 6.5). The suspended bacterial cells were
disrupted by an UH-50 type ultrasonic homogenizer (manufactured by SMT)
and thereafter bacterial debris was centrifugally removed to obtain a
cell-free extract. The reduction activity to 3-oxo-3-phenylpropanenitrile
and GDH activity of the cell-free extract were measured and expressed as
relative activities in Table 1. In each of the two types of transformants
obtained in Example 5, the reduction activity to
3-oxo-3-phenylpropanenitrile was observed. Furthermore, the expression of
the GDH activity was observed in E. coli HB101 (pNTCMG1) containing a GDH
gene. The reduction activity to 3-oxo-3-phenylpropanenitrile was measured
by the method described in Example 1. The GDH activity was obtained by
adding 0.1 M glucose, 2 mM coenzyme NADP and a crude enzyme solution to
1M tris hydrochloride buffer (pH 8.0), performing a reaction at
25° C. for one minute, measuring absorbance at a wave length of
340 nm, calculating an increasing rate of the absorbance. The enzymatic
activity for reducing 1 μmol of NADP to NADPH per minute in the
reaction conditions was defined as 1 unit. The protein concentration of
the cell-free extract was measured by a protein assay kit (manufactured
by BIO-RAD).

[0118]To 50 ml of the cell-free extract of E. coli HB101 (pNTCM) prepared
in the same manner as in Example 6, 2000U of glucose dehydrogenase
(manufactured by Amano Enzyme Inc.), 3 g of glucose, 6 mg of NADP and 2 g
of 3-oxo-3-phenylpropanenitrile were added. The mixture was stirred at
30° C. for 22 hours while adjusting the pH of the mixture to 6.5
by adding 5 M sodium hydroxide dropwise. After completion of the
reaction, extraction was performed with ethyl acetate. The solvent was
removed and then an extracted material was analyzed. As a result,
(R)-3-hydroxy-3-phenylpropanenitrile having an optical purity of 97.3%
e.e. was obtained in a yield of 86.3%.

[0120]To 50 ml of the cell-free extract of E. coli HB101 (pNTCMG1)
prepared in the same manner as in Example 6, 3 g of glucose, 6 mg of NADP
and 2 g of 3-oxo-3-phenylpropanenitrile were added. The mixture was
stirred at 30° C. for 22 hours while adjusting the pH of the
mixture to 6.5 by adding 5 M sodium hydroxide dropwise. After completion
of the reaction, extraction was performed with ethyl acetate. The solvent
was removed and then an extracted material was analyzed. As a result,
(R)-3-hydroxy-3-phenylpropanenitrile having an optical purity of 97.4%
e.e. was obtained in a yield of 92.1%.

[0121]The amounts of 3-oxo-3-phenylpropanenitrile and
(R)-3-hydroxy-3-phenylpropanenitrile and the optical purity of
(R)-3-hydroxy-3-phenylpropanenitrile were obtained in the same manner as
in Example 7.

[0122]Production of (R)-2-chloro-1-(3'-chlorophenyl)ethanol Using
Transformant

[0123]To 50 ml of a culture solution of E. coli. HB101 (pNTCMG1) prepared
in the same manner as in Example 6, 5 g of glucose, 6 mg of NADP, 4 g of
2-chloro-1-(3'-chlorophenyl)ethanone and 4 g of toluene were added. The
mixture was stirred at 30° C. for 24 hours while adjusting the pH
of the mixture to 6.5 by adding 5 M sodium hydroxide dropwise. After
completion of the reaction, the reaction solution was extracted with
toluene. The solvent was removed and then an extracted material was
analyzed. As a result, (R)-2-chloro-1-(3'-chlorophenyl)ethanol having an
optical purity of 82.0% e.e. was obtained in a yield of 90.5%.

[0125]To 50 ml of a culture solution of E. coli HB101 (pNTCMG1) prepared
in the same manner as in Example 6, 6 g of glucose, 4 mg of NADP were
added. The reaction solution was stirred at 30° C. While the pH of
the reaction solution was adjusted to pH 6.5 by adding 5 M sodium
hydroxide dropwise, 5 g in total of ethyl 4-chloro-3-oxobutyrate was
added at a rate of 0.5 g/hour. Stirring was continued for a further 5
hours. After completion of the reaction, the reaction solution was
extracted with ethyl acetate. The solvent was removed and then an
extracted material was analyzed. As a result, ethyl
(S)-4-chloro-3-hydroxybutyrate having an optical purity of 80.5% e.e. was
obtained in a yield of 93.1%.

Production of tert-butyl 6-benzoyloxy-3,5-dihydroxyhexanoate Using
Transformant

[0127]To 40 ml of a culture solution of E. coli HB101 (pNTCMG1) prepared
in the same manner as in Example 6, 3 g of glucose, 6 mg of NADP, 4 g of
tert-butyl (S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate and 1.6 g of toluene
were added. The reaction solution was stirred at 30° C. for 24
hours while adjusting the pH of the reaction solution to pH 6.5 by adding
5 M sodium hydroxide dropwise. After completion of the reaction, the
reaction solution was extracted with toluene. The solvent was removed and
then an extracted material was analyzed. As a result, tert-butyl
(3R,5R)-6-benzyloxy-3,5-dihydroxyhexanoate having an excess rate of a
diastereomer of 99.7% d.e. was obtained in a yield of 92.3%.

[0129]To 100 mM phosphate buffer (pH 6.5) containing 0.33% (v/v)
dimethylsulfoxide, a substrate, a carbonyl compound serving as a
substrate was dissolved so as to obtain a final concentration of 1 mM and
a coenzyme NADPH in a final concentration of 0.25 mM. To the mixture, the
purified polypeptide prepared in Example 1 was added in an appropriate
amount and a reaction was performed at 30° C. for one minute.
Based on a reduction rate of absorbance of the reaction solution at a
wavelength of 340 nm, the reduction activity to each carbonyl compound
was calculated. This was expressed by a relative value to the activity to
3-oxo-3-phenylpropanenitrile as being in Table 2. As is apparent from
Table 2, the polypeptide of the present invention exhibits reduction
activity to a wide variety of carbonyl compounds.